QUESTION

DYSFUNCTIONALPROTEIN DEGRADATION

NEURODEGENERATION

?

NEURODEGENERATION

associated with

Alzheimer’s DiseaseParkinson’s Disease

Huntington’s DiseaseAmyotrophic Lateral

Sclerosis

Parkinson’s Disease

Alzheimer’s Disease

Huntington’s Disease

Amyotrophic lateral sclerosis

Spinocerebellar Ataxia

Disease

accumulation of misfolded proteins

Cell Death

Neurodegenerative Neurodegenerative DisordersDisorders

Ubiquitin-Protein Aggregates

HUNTINGTON’S ALZHEIMER’S

PARKINSON’S LOU GEHRIG’S

f

PD: ubiquitin

c

AD: tau

d

AD: ubiquitin

Protein DegradationProtein DegradationTurnover of protein is NOT constantTurnover of protein is NOT constant

Half lives of proteins vary from minutes to infinityHalf lives of proteins vary from minutes to infinity

““Normal” proteins – 100-200 hrsNormal” proteins – 100-200 hrs

Short-lived proteinsShort-lived proteinsregulatory proteinsregulatory proteins

enzymes that catalyze committed stepsenzymes that catalyze committed stepstranscription factotstranscription factots

Long-lived proteinsLong-lived proteinsSpecial cases (dentin, crystallins)Special cases (dentin, crystallins)

Protein Degradation

ENZYME half-lifeOrnithine decarboxylase 11

minutes-Aminolevulinate synthetase 70

minutesCatalase 1.4

daysTyrosine aminotransferase 1.5

hoursTryptophan oxygenase 2 hoursGlucokinase 1.2

daysLactic dehydrogenase 16 daysHMG CoA reductase 3 hour

Proteins are not degraded at the same rate

Protein Degradation

Example: Lactic Acid DehydrogenaseTissue Half-lifeHeart 1.6

daysMuscle 31 daysLiver 16

days

• May depend on tissue distribution

• Protein degradation is a regulated processExample: Acetyl CoA carboxylase

Nutritional state Half-lifeFed

48 hoursFasted 18

hours

Protein Degradation Ubiquitin/Proteasome Pathway

80-90%Most intracellular proteins

• Lysosomal processes10-20%

Extracellular proteinsCell organellesSome intracellular proteins

Two Sites for Protein Degradation

Proteasomes

Large (26S) multiprotein complex (28 subunits)

Degrades ubiquitinated proteins

Lysosomes

Basal degradation – non-selective

Degradation under starvation – selective for “KFERQ” proteins

The Ubiquitin/ProteasomeThe Ubiquitin/Proteasome

PATHWAYPATHWAY

UBIQUITIN

KK

GG

Small peptide that is a “TAG” 76 amino acids C-terminal glycine - isopeptide

bond with the -amino group of lysine residues on the substrate

Attached as monoubiquitin or polyubiquitin chains

Three genes in humans:

Two are stress genes (B and C)

One, UbA as a fusion protein

Tetra-Ubiquitin

Cook, W.J. et al. (1994) J. Mol. Biol. 236, 601-609

UBIQUITIN GENES

Degradation by the 26S PROTEASOME

Ubiquitin/Proteasome Pathway

UbiquitinationUbiquitination

The Ubiquitin/Proteasome Pathway

Four Main Steps:

UBIQUITINATIONUBIQUITINATION

RECOGNITIONRECOGNITION

DEGRADATIONDEGRADATION

DEUBIQUITINATIONDEUBIQUITINATION

UBIQUITINATED PROTEINSUBIQUITINATED PROTEINS

UBIQUITIN UBIQUITIN CHAINSCHAINS

MQIFVKTLTGKTITLEVESSDTIDNVKAKIQDKEGIPPDQ

QRLIFAGKQLEDGRTLADYNIQKESTLHLVLRLRGG

6 11 29

48 63

27 33

Functions of Functions of UbiquitinationUbiquitination

• Poly-ubiquitinationPoly-ubiquitination Targets proteins from Targets proteins from Cytoplasm, Nuclear & ER for Cytoplasm, Nuclear & ER for degradation by the degradation by the PROTEASOME PROTEASOME

DNA repairDNA repair

•Mono-ubiquitinationMono-ubiquitination Receptor internalizationReceptor internalization Endocytosis – lysosomeEndocytosis – lysosome Transcription regulationTranscription regulation

Ubiquitination of proteins is a FOUR-step process

First, Ubiquitin is activated by forming a link to “enzyme 1” (E1).

Then, ubiquitin is transferred to one of several types of “enzyme 2” (E2).

Then, “enzyme 3” (E3) catalizes the transfer of ubiquitin from E2 to a Lys -amino group of the “condemned” protein.

Lastly, molecules of Ubiquitin are commonly conjugated to the protein to be degraded by E3s & E4s

AMP

UBIQUITIN ACTIVATIONUBIQUITIN ACTIVATION

E1E1

UBIQUITIN UBIQUITIN ADENYLATEADENYLATE

THIOLTHIOLESTERESTER

E1-s-co-Ub + E2-SH -----> -----> E2-s-co-Ub + E1

UBIQUITIN CONJUGATION

CLASS 1 – UBC domains only; require E3s for Ub; target substrates for degradation

CLASS 2 – UBC domains & C-terminal extensions; UBC2 = RAD6 – DNA repair not degradation; no E3s

CLASS 3 – UBC domains & N-terminal extensions; function not known

UBC domainN C

E2-s-co-Ub + Protein-NHE2-s-co-Ub + Protein-NH22 -------> ------->

E2-SH + Protein-NH-CO-UbE2-SH + Protein-NH-CO-Ub

(ubiquitin = polyubiquitin (ubiquitin = polyubiquitin chains)chains)

UBIQUITIN LIGATIONUBIQUITIN LIGATION E3E3

““recogninsrecognins” =” = recognize recognize a motifa motif (DEGRON)(DEGRON) on a on a

protein substrateprotein substrate

Three Major Classes of E3

3) multi-subunit cullin based E3s

1) HECT-domain E3s

2) RING finger-domain E3s

Ubiquitin Ligases (E3)1) HECT-domain containing a conserved Cys

2) RING finger-domain Cys & His residues are ligands to two Zn++ ionsstabilizes a molecular scaffold

Ubiquitin Ligases (E3) (cont.)

3) Complex E3s: Multiple subunitsEx: SCF-type E3, VBC-Cul2 E3 and other cullin based E3s, Anaphase promoting complex (APC)

-they provide a Scaffold for Ub transfer

-F-box – substraterecognition

ELONGATION = E4

1)U box = CHIP (+parkin)

2)Non-U box = p300 (p53)

3)E3-E4 complex = C. elegans

ACTIVATION OF A UBIQUITIN-LIGASEACTIVATION OF A UBIQUITIN-LIGASE

DEGRADATION SIGNALSDEGRADATION SIGNALSsubstratessubstrates

RECOGNITIONRECOGNITION

N-end RULE

N-end RULEN-degron - signal N-recognin - E3

DEGRADATIONDEGRADATION

PROTEASOME COMPONENTS

19-3

20S Proteaso

me

19S Particle

26S Proteasome

ATP

The 26S proteasomeThe 26S proteasome

Ubiquitinated proteins are degraded by the proteasome

Proteasomal protein degradation consumes ATP.

The proteasome degrades the proteins to ~8 amino-acid peptides.

Access of proteins into the proteasome is tightly regulated.

The peptides resulting from the proteasome activity diffuse out of the proteasome freely.

Ubiquitinated proteins are degraded in the cytoplasm and nucleus by the proteasome.

Hydrolysis peptide bonds after:Hydrolysis peptide bonds after:

hydrophobic a.a.hydrophobic a.a. = = CHYMOTRYPSIN-CHYMOTRYPSIN-LIKE - LIKE - 55

acidic a.a.acidic a.a. = (-) = (-)CASPASE-LIKE CASPASE-LIKE --11

basic a.a.basic a.a. = (+) = (+)TRYPSIN-LIKE TRYPSIN-LIKE --22

DEUBIQUITINATIONDEUBIQUITINATION

De-ubiquitinatingDe-ubiquitinating

Ubiquitin – like Ubiquitin – like proteinsproteins““UBP”UBP”

SmallUbiquitin-like

Modifier

Ubiquitin – like Ubiquitin – like modifiersmodifiers

LYSOSOMESLYSOSOMES

Digestive System of the Digestive System of the CellCell

• DigestsDigests – ingested materials– obsolete cell components

• DegradesDegrades macromolecules of all types

– Proteins– Nucleic acids– Carbohydrates– Lipids

• HeterogeneHeterogeneousous

Lysosomal Lysosomal EnzymesEnzymes

• 50 different degradative enzymes

• Acid hydrolasesAcid hydrolases– Active at pH 5

(inside lysosome)– Inactive if released

into cytosol (pH 7.2)

• Acidic pHAcidic pH of lysosomes maintained by a proton

pump in the lysosomal

membrane– Requires ATP, thus mitochondria

Different pathways lead to the lysosome 1) Phagocytosis–Cell “eating” of material

> 250nm

2) Pinocytosis–Cell “drinking” < 150nm

3) Receptor Mediated Endocytosis-clathrin-coated pits

4) Autophagy–“self eat” of old worn out organelles, – important in cell degradation during apoptosis

Protein degradation in the Protein degradation in the lysosomeslysosomes

Lysosomes degrade extracellular proteins that the cell incorporates by endocytosis.

Lysosomes can also degrade intracellular proteins that are enclosed in other membrane-limited organellas.

In well-nourished cells, lysosomal protein degradation is non-selective (non-regulated).

In starved cells, lysosomes degrade preferentially proteins containing a KFERQKFERQ “signal” peptide.

The regression of the uterus after childbirth is mediated largely by lysosomal protein degradation

AUTOPHAGYAUTOPHAGY

- Macroautophagy – inducible (mTOR)- Macroautophagy – inducible (mTOR)(autophagy)(autophagy)

- Microautophagy - constitutive- Microautophagy - constitutive

- Chaperone-mediated autophagy - Chaperone-mediated autophagy (CMA) – KFERQ motif(CMA) – KFERQ motif

AUTOPHAGY

AUTOPHAGY (MACRO) PATHWAYOxidative stress Infection Protein aggregates

AUTOPHAGY (MACRO) PATHWAY

Lysosome

AUTOPHAGY PATHWAY17 genes = Atg

Stress

TORtarget of Rapamycin

Tight association(negative regulation)

1) INDUCTION

AUTOPHAGY PATHWAY

2) AUTOPHAGOSOME FORMATION

NEW MEMBRANE = ER

LIPID KINASES (PHOSPHATIDYL

INOSITOL) SIGNALING COMPLEX

AUTOPHAGY PATHWAY

PROTEIN CONJUGATION SYSTEM~ TO THE UBIQUITIN SYSTEM

3) DOCKING & FUSION

DIM

ERIZ

ATIO

N

MEMBRANE ASSOCIATION

phosphatidylethanolamine

AUTOPHAGY PATHWAY

4) BREAKDOWN

RECYCLING &

RETRIEVAL

Only Atg19 & Atg8-PEremain associated with the

Autophagosome;Others are re-cycled

AUTOPHAGY PATHWAY

Neurodegenerative diseasesNeurodegenerative diseases- PD, AD, HD and TSE- aggregate removal

Infectious diseasesInfectious diseases- remove pathogens

CancerCancer- sequester damaged organelles- promote autophagic death

80-90%

26S PROTEASOME

10-20%

AUTOPHAGY/LYSOSOMEPATHWAY

Degradation of Proteins

Why? 26S Proteasome

AGGREGATES

Environmental &Genetic InsultsInflammation

Aging

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ub

Ubiquitin-Protein Aggregates

Lysosome

26S Proteasome

?

SUBSTRATEUb

Ub UbUb

CROSS-TALK

AUTOPHAGYINDUCTION

The End

AUTOPHAGY & UPPHuman neuroblastoma SK-N-SH cells

&Rat Spinal Cord Organotypic Cultures

Model for ALS

CELL RECOVERYCELL RECOVERY AUTOPHAGYAUTOPHAGY

UPP UPP

CELL DEATH

ENVIRONMENTAL & ENVIRONMENTAL & GENETIC INSULTSGENETIC INSULTS

ProteinProteinAggregatAggregat

eses

Other ?Other ?

? Protein Protein

DegradationDegradation

p62/SQSTM1HDAC6

Neurodegeneration = ubiquitin inclusions

HUNTINGTON’S Disease

ALZHEIMER’S Disease

PARKINSON’S DiseaseAmyotrophic Lateral

Sclerosis

f

PD: ubiquitin

c

AD: tau

d

AD: ubiquitin